Disorders of abnormal cell proliferation are characterized by inappropriate growth or multiplication of one or more cell types. They include malignant (i.e., cancer) as well as non-malignant disorders. Many of these diseases also include an inflammatory component. Psoriasis represents one type of non-malignant disorder of abnormal cell proliferation. The disorder is characterized by psoriatic skin plaques representing highly localized sites of deregulated growth and inflammation. While the cause of psoriasis is poorly understood, it is thought to involve both a genetic and environmental component. Moderate-to-severe psoriasis has traditionally been treated with systemic therapies such as cyclosporine, methotrexate, retinoids, and phototherapy (i.e., ultraviolet B, psoralen plus ultraviolet A). Traditional treatments, however, suffer limitations including significant side effects, lack of durable efficacy, and inconvenient administration schedules.
Uncontrolled or inappropriate chronic inflammatory responses are characteristic of a variety of diseases and disorders. Inflammatory bowel disease (IBD), including both Crohn's disease and ulcerative colitis, provides one example of an inflammatory disorder. IBD affects the quality of life of more than one million Americans. At present, aminosalicylates (5-ASA), corticosteroids, immune modifiers and antibiotics are used to treat Crohn's disease. Current therapies, however, are ineffective in many patients and present significant side effects including slow onset of action and toxicity.
Antifolates are compounds that interfere with various stages of foliate metabolism. An intact foliate enzyme pathway is important to maintain de novo synthesis of the building blocks of DNA, as well as of the important amino acids. Antifolate targets include the various enzymes involved in foliate metabolism, including (i) dihydrofolate reductase (DHFR); (ii) thymidylate synthase (TS); (iii) folylpolyglutamyl synthase; and (iv) glycinamide ribonucleotide (GAR) and aminoimidazole carboxamide ribonucleotide (AICAR) transformylases.
Antifolates are folate acid analogs. For a general review of antifolates, see Montgomery JA and Piper Jr. Design and Synthesis of Folate Analogs as Antimetabolites. In Folate Antagonists as Therapeutic Agents. Volume 1: Biochemistry, Molecular Actions and Synthetic Design. Eds. Sirotnak F M, Burchall J J, Ensminger W D and Montgomery J A. Academic Press. pp 219-261, 1984; Thomas W. Current Oncology Reports (2003) 5:114-125; Graffner NM. Approaches to Soft Drug Analogues of Dihydrofolate Reductase Inhibitors, in Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 252 (2001); Beale, P., and Clarke, S. Tomudex. Clinical development. In: A. L. Jackman (ed.), Antifolate Drugs in Cancer Therapy, pp. 167-191. Allegra C J: Antifolates, in Chabner B A, Collins J M (eds): Cancer Chemotherapy: Principles & Practice, pp 110-153. Philadelphia, Lippincott, 1990.
Folic acid contains a pteridine ring, para-aminobenzoic acid and a glutamate residue.

Methotrexate, a DHFR inhibitor, is among the earliest antifolates. It is a classical antifolate, meaning it is characterized by a p-aminobenzogylglutamic acid side chain, and closely resembles the folic acid molecule. MTX differs from folic acid by the substitution of an amino group for a hydroxyl at the 4-position of the pterdine ring and by the methylation of the amine of the para-aminobenzoic moeity. The substitution of an amino group for a hydroxyl at the 4-position of the pterdine ring changes the enzyme substrate into a tight binding inhibitor of DHFR.

Both MTX and naturally occurring folate compounds undergo intracellular metabolism to polyglutamate derivatives. This polyglutamylation is catalyzed by the enzyme folylpolyglutamyl synthase (FPGS), which attaches up to six glutamate residues to the molecule, which helps to trap it within the cell. Polyglutamylation of MTX occurs more slowly compared to naturally occurring antifolates, but the resulting methotrexate polyglutamylates have extremely long intracellular half-lives, and can be detected in some tissues more than several months after a single drug administration (Takimoto C H et al. Oncology (1995) 9(7): 649-656).
The most common use of MTX is as an anti-cancer drug. MTX is curative of choriocarcinoma and Burkett's lymphoma. It has also widely used as a single agent or in combination with other drugs for the treatment of various forms of human cancer. More recently, MTX has been shown to have anti-inflammatory and immunosuppressive properties with accompanying activity against autoimmune disorders. MTX is now widely prescribed as an immunosuppressive agent in the treatment of autoimmune diseases, including rheumatoid arthritis (Weinblatt M E et al. N. Ensl. J. Med. (1985) 312:818; Wilke W S (Ed). Methotrexate Therapy in Rheumatic Disease. Marcel Dekker, Inc. (1989). Intrinsic and acquired resistance to MTX and other antifolate analogues limits their clinical effectiveness, however. Apart from resistance, major limitations of MTX treatment include bone marrow toxicity, gastrointestinal ulceration and liver and kidney damage.
A number of antifolates have been designed to overcome these limitations. Rational design has focused, for example, on the development of antifolates with greater lipid solubility and/or improved transport characteristics relative to methotrexate (Takimoto C H et al. Oncology (1995) 9(7); 649-656). Representative non-classical agents include trimetrexate and piritrexim (Kamen B A et al. J. Biochem. Pharmacol. (1984) 33: 1697-1984; Duch D S et al. Cancer Res. (1982) 42: 3987-3994). Unlike classical antifolates, non-classical antifolates lack the glutamate moiety, and therefore do not require carrier-mediated cellular uptake. These lipophilic antifolates are used against opportunistic infections (e.g., Pneumocystic carinii pneumonia, PCP) in individuals with AIDS and other disorders of the immune system and have undergone extensive clinical testing as anticancer agents.

Other antifolates in clinical development specifically target folate-dependent enzymes such as TS or GARFT, thereby directly affecting pools of nucleotides available for DNA synthesis (Takemura Y. et al. Anti-Cancer Drugs (1997) 8: 3-16; Habeck L L et al. Cancer Res (1994) 54: 1021-1026). Direct and specific TS inhibitors have been studied as potential anticancer drugs (Stout T J et al. Biochemistry (1999) 38: 1607-1617). Of these, Tomudex (raltitrexed; ZD1649), [N-{5-[N-(3-,4-dihydro-2-methyl-4-oxoquinazoline-6-yl-methyl)-N-methylamine]-2-theroyl}-L-glutei acid], is one of the most extensively evaluated and has been approved for treatment in Europe (Van Custom Euro. J. Cancer, (1999) 35(Suppl.1): 1-2; Jack man AL. Invest. New Drugs, (1996) 14: 305-316). Comdex undergoes substantial polyglutamylation within the cell. Comdex and its polyglutamates do not appear to inhibit DHFR, GAR or AICAR transformylase, suggesting that the drug is a pure TS inhibitor.

Inhibitors of glycinamide ribotide formyltransferase (GARFT) have also been developed. Lometrexol ((5,10-dideazatetrahydrofolate [DDATHF]) is a specific GARFT inhibitor that has shown anti-tumor properties (Habeck L L et al. Cancer Res. (1994) 54: 1021-1026). Early clinical trials, however, were confounded by cumulative myelosuppression that prevented repetitive administration (Roberts J D. Cancer Chemother Pharmacol. (2000) 45(2):103-10). LY309887 (6R-2′,5′-thienyl-5,10-dideazatetrahydrofolic acid) is a thiophene analogue of lometrexol, is a second generation GARFT inhibitor (Mendelsohn L G. Investig. New Drugs (1996) 14: 287-294).

In 1991, Nair et al. demonstrated that contrary to the widely accepted notion, polyglutamylation of classical antifolates is not essential for anti-tumor activity and further, that this metabolic transformation is actually undesirable because it may cause the loss of pharmacological control and target specificity of the drug (Nair M G et al. J. Med. Chem (1991) 34: 222-227). This new finding let to the discovery of a number of nonpolyglutamylatable classical antifolates (Nair M G et al. Proc. Amer. Assoc. Cancer. Research. (1998) 39:431).
U.S. Pat. No. 5,073,554 (Nair) describes methylene-1-deazaaminopterine (MDAM), a nonpolyglutamylatable antifolate compound. MDAM has been developed as an experimental anticancer drug for the treatment of human solid tumors (Cao S. et al. Clinical Cancer Research (1996) 2(4): 707-712); Johansen M et al. Cancer Chemother Pharmacol. (2004) 53(5):370-6). U.S. Pat. No. 5,550,128 (Nair et al) describes the active enantiomer of MDAM as the one possessing the L-configuration.

Further investigation by Nair et al. of the metabolic disposition of certain non-polyglutamylatable antifolates led to the unexpected finding that the presence of the 4-methyleneglutamate moiety modulates the binding of such compounds to the liver enzyme aldehyde oxidase, which mediates their oxidative deactivation to the corresponding 7-hydroxy derivatives (Cellular. Pharmacology (1996) 3: 29). U.S. Pat. No. 5,912,251 (Nair) describes metabolically inert classical antifolates, including 4-Amino4-deoxy-5,8,10-trideazapteroyl-4′-methylene glutamic acid, which are non-polyglutamylatable and non-hydroxylatable. They are said to be useful in the treatment of neoplastic disease (leukemia, ascetic and solid tumors), asthma and related anti-inflammatory disease, and for the treatment of rheumatoid arthritis and other autoimmune diseases.
wherein X is CH2, CHCH3, CH(CH2CH3), NH, or NCH3.
wherein X is CH2.